CN112522155B

CN112522155B - Bacillus licheniformis and application thereof

Info

Publication number: CN112522155B
Application number: CN202011497324.2A
Authority: CN
Inventors: 张蔚; 郭伟伟; 罗远恒; 高俐; 张正瀚
Original assignee: Shanghai Shenglong Environmental Remediation Materials Co ltd
Current assignee: Shanghai Shenglong Environmental Technology Co.,Ltd.
Priority date: 2020-11-17
Filing date: 2020-12-17
Publication date: 2021-09-21
Anticipated expiration: 2040-12-17
Also published as: CN112522155A

Abstract

The invention provides a new application of Bacillus licheniformis. The oxygen-containing group has the ability to adsorb heavy metals. The bacillus licheniformis which is numbered as bacillus licheniformis and produces pectinase is used for fermenting peanut shells, so that pectin on the peanut shells is degraded, oxygen-containing groups which are coated on cellulose and hemicellulose and have heavy metal adsorption capacity are removed, and the restriction is relieved. In addition, metabolites with oxygen-containing groups are generated in the microbial culture process, and meanwhile, the peanut shell structure is looser due to pectin hydrolysis, so that the adsorption surface area is increased, and the adsorption capacity is improved by multiple factors. Thereby effectively improving the adsorption capacity of the peanut shells to heavy metals.

Description

Bacillus licheniformis and application thereof

Technical Field

The invention belongs to the field of biotechnology, and relates to fermentation engineering and enzyme engineering.

Background

With the increasingly accelerated industrial development, the heavy metal ion pollution to the land is also becoming more serious, and at present, two main approaches are available for treating the heavy metal pollution:

1. the existence state of heavy metals is changed, the activity of the heavy metals is reduced, the heavy metals are passivated, and are separated from food chains, so that the toxicity of the heavy metals is reduced;

2. the method comprises the steps of absorbing heavy metals in soil by using special plants, removing the plants or changing the heavy metals into soluble state and free state by using an engineering technology, leaching, and collecting the heavy metals in leacheate, thereby achieving the dual purposes of recovering the heavy metals and reducing the heavy metals in the soil.

The first route is usually used because the latter route is more costly and longer in cycle time than the other route.

The method for changing the existing state of heavy metal comprises physical method, chemical method and biological method. Wherein, the physical method and the chemical method have large processing engineering quantity and high cost. The biological method comprises an animal method, a plant method and a microbiological method, wherein the microbiological method has the advantages of rich resources, short period, no influence on cultivation, various comprehensive treatment effects and the like.

The cellulose molecular structure contains more oxygen-containing groups such as hydroxyl, carboxyl and the like, and has the capacity of adsorbing heavy metals. Peanut shells are renewable biomass, and have the function of adsorbing heavy metals because the peanut shells are rich in cellulose. But because the cellulose in the peanut shells is encapsulated by pectin, the oxygen-containing groups are bound. Thus, unprocessed peanut shells have a low adsorption capacity. The biological activated carbon is generally prepared into biological activated carbon and is popularized and applied as an adsorption material. However, the preparation of the biological activated carbon by using the peanut shells has harsh requirements, high manufacturing cost and high product price.

Disclosure of Invention

The invention aims to provide a new application of bacillus licheniformis to solve the problems in the prior art.

The invention is realized by the following technical scheme:

the Bacillus licheniformis (Bacillus licheniformis) is preserved in China general microbiological culture collection management center with the preservation number of CGMCC No.20977, the preservation address is China academy of sciences microbiology No. 3 of Beijing republic of Naja district, Xilu No. 1 of Beichen province, and the preservation date is 10 months and 29 days in 2020.

The strain is separated and screened from saline-alkali soil, and is sent to the institute of microorganisms of Chinese academy of sciences to be identified as bacillus licheniformis. The test tube slant seeds are prepared by the applicant and are placed in a refrigerator for refrigeration at about 4 ℃.

Use of a bacillus licheniformis as described above in the production of alkaline pectinase.

A biological agent, the active ingredients of which comprise:

such as the aforementioned bacillus licheniformis, or/and,

such as the fermentation product of Bacillus licheniformis as described previously.

A method for preparing the biological agent, which comprises the following steps:

inoculating the bacillus licheniformis into a fermentation culture medium for fermentation; the fermentation medium comprises peanut shells.

Preferably, the preparation method of the fermentation medium comprises the following steps:

dissolving edible alkali, dipotassium hydrogen phosphate and magnesium sulfate in water to obtain a nutrient solution;

and (3) crushing the peanut shells, adding the crushed peanut shells into the nutrient solution, and uniformly dispersing the crushed peanut shells.

Preferably, the fermentation temperature is 30-45 ℃.

Preferably, the edible alkali, the dipotassium hydrogen phosphate and the magnesium sulfate are respectively 2%, 0.1% and 0.02% in the nutrient solution, and the weight ratio of the peanut shells to the nutrient solution is 1: (1-1.2).

Preferably, the ratio of the bacillus licheniformis to the fermentation medium is as follows: 1-3 eggplant bottle seeds are inoculated into every 10kg of peanut shells.

Preferably, the inoculation amount of the solid seed koji is 10-20% of the weight of the peanut shells.

Use of a biological agent as described above in the treatment of heavy metal ions in soil.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention utilizes biotechnology to partially hydrolyze pectin in peanut shells, so that more oxygen-containing groups (such as hydroxyl, carboxyl, carbonyl and the like) are exposed; moreover, the aldehyde group and carboxyl group generated by pectin hydrolysis are increased; and organic acid generated by microbial metabolism, and simultaneously the structure of the peanut shell becomes loose, so that the specific surface area of the peanut shell is increased, namely the contact area of the oxygen-containing chemical group and the adsorbed material is increased, and the adsorption capacity of the peanut shell on heavy metal is improved from multiple aspects;

2. the method has the advantages of simple operation, less equipment investment, wide raw material source, low price, waste utilization, reduction of incineration pollution and contribution to popularization.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a photograph of a slant of a strain of Bacillus licheniformis of the present invention and a seed in an eggplant bottle;

FIG. 2 is a photograph of the biological preparation obtained in the present invention.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

EXAMPLE 1 this example relates to the expanded culture of Bacillus licheniformis

First, the strain is enlarged and cultured

1. And (3) strain preservation: and (5) preserving the test tube by using a slant surface.

2. Slant seed culture

1) Preparing a slant culture medium: after starch, corn steep liquor, dipotassium hydrogen phosphate and magnesium sulfate are dissolved in water, the mass fractions of the starch, the corn steep liquor, the dipotassium hydrogen phosphate and the magnesium sulfate are respectively controlled as follows: 1%, 2%, 0.1%, 0.02%, adjusting pH to about 7 with hydrogen hydroxide, adding agar 2.5% of the solution, sterilizing at 121 deg.C for 30 min. Preparing sodium carbonate into an aqueous solution with the mass fraction of 20%, sterilizing at 121 ℃ for 30 minutes, uniformly mixing the sodium carbonate and other culture medium components according to the dosage of the final concentration of 1% while the mixture is hot, subpackaging the mixture into sterilized sterile test tubes, and placing the sterilized test tubes into an inclined plane. After the slant is solidified, placing the solidified slant in an incubator, carrying out blank culture for 2 days at 37 ℃, checking the sterility, and then placing the solidified slant in a refrigerator for refrigeration at about 4 ℃ for standby.

2) Inoculating and culturing slant seeds: taking a blank inclined plane, inoculating seeds on the original inclined plane in a sterile room according to a conventional method, and then placing the inoculated seeds in an incubator to culture for 5-7 days at 30 ℃. The seeds are refrigerated in a refrigerator at about 4 ℃ as the seeds with the inclined surfaces.

3) Slant strain activation: before use, the slant seeds are transferred to a blank slant according to a conventional method, and are placed in an incubator at 30 ℃ for 2 days to be activated. The liquid seed is then transferred. As shown in fig. 1.

3. Liquid seed culture:

1) liquid seed culture medium: 1g of starch, 2g of corn steep liquor, 0.1g of dipotassium phosphate, 0.02g of magnesium sulfate and 100mL of tap water are heated and dissolved, the pH value is adjusted to about 7 by using 2mol/L of sodium hydroxide, 50mL of 250mL triangular bottled culture solution is sterilized at 121 ℃ for 30 minutes.

2) Inoculation: 2.5mL of a 20% sodium carbonate solution that had been sterilized was added before inoculation of the liquid seed medium. Shaking up. Inoculating activated slant strain 1 ring with inoculating loop.

3) Liquid seed culture: after the liquid seed culture medium is inoculated, the liquid seed culture medium is placed in a shaking table and cultured for 24 hours at 180rpm and 30-35 ℃.

4. Culturing eggplant bottle seeds:

1) eggplant bottle inclined plane solid medium: the formula of the culture medium is the same as that of the liquid seed culture medium. Each eggplant bottle is filled with 80mL of liquid seed culture medium containing other components except sodium carbonate, then 2g of agar is added, a cotton plug is added, the mixture is sterilized at 121 ℃ for 30 minutes, 4mL of 20% sodium carbonate solution sterilized in the same way is added while the mixture is hot, the mixture is shaken evenly and placed on a slope. After the slant solidification, the culture was carried out at 35 ℃ for 2 days in a blank, and sterility was confirmed. Placing in a refrigerator, and refrigerating at about 4 deg.C for use.

2) Eggplant bottle inoculation: sucking 0.1mL of liquid seed liquid by using a sterile pipette; adding onto the slant of eggplant bottle, and uniformly coating the liquid seed on the slant with sterile scraper.

3) Culturing eggplant bottle seeds: and (4) placing the inoculated eggplant bottle inclined plane into an incubator to be cultured for 2-3 days at the temperature of 30-37 ℃. As seeds of a solid seed koji.

Embodiment 2 this embodiment relates to a method for preparing a solid seed koji, which specifically includes the following steps:

1. culturing solid seed koji:

1) the components of the culture medium:

A. the nutrient solution formula comprises: 2% of sodium carbonate, 0.1% of dipotassium hydrogen phosphate (chemical fertilizer), 0.02% of magnesium sulfate (chemical purity), and is prepared by tap water. Stirring to dissolve.

B. Solid seed koji burdening: crushing peanut shells, and adding nutrient solution according to the proportion of 1: 1.2. Turning and stirring uniformly in a stainless steel basin. Sterilizing at 121 deg.C for 30 min. Namely the solid seed koji culture medium.

2) Inoculating a seed koji: and (3) inoculating the sterilized starter culture medium with 1-3 eggplant bottle seeds in a starter culture room after the temperature of the material is cooled to 37-40 ℃ according to 10kg of peanut shells. 50mL of sterile water was added to each eggplant bottle seed, and the bacterial cells were scraped off with a sterile spatula and carefully stirred up. Separating a small amount of material from the edge of the material, adding eggplant bottle slant seeds (optionally adding culture medium in the eggplant bottle slant seeds), and stirring. Then the mixture is stirred evenly with the rest materials.

3) The seed koji culture method comprises the following steps: the seed koji can be cultured in a stainless steel basin, a tray or a koji culture bed, the materials are firstly accumulated after being inoculated, a thermometer is inserted for covering and heat preservation culture, and when the temperature of the culture materials rises to 35-40 ℃, the culture materials can be raked; controlling the thickness of the material layer to be about 10-20 cm; and (3) covering and preserving heat, continuing culturing, controlling the temperature to be 30-40 ℃ in the culturing process, intermittently turning and stirring for cooling according to temperature change, and keeping the temperature of the culture material in the culture room in summer and in winter. Culturing the koji for 3-5 days to obtain solid seed koji,

the Bacillus licheniformis strain is enlarged and cultured to prepare a solid seed koji in the examples 1 and 2. Used as the seed of the peanut shell solid fermentation.

Example 3 this example relates to peanut shell fermentation to produce a peanut shell preparation

1. Fermentation and batching: 100kg of crushed peanut shells are weighed.

2.4kg of sodium carbonate, 120g of dipotassium hydrogen phosphate and 24g of magnesium sulfate are weighed, and 120kg of tap water is added. Stirring to dissolve. Adding into peanut shell, and stirring. And then inoculation can be carried out.

2. Inoculating a fermentation material: mixing the culture materials, weighing 10kg of solid seed yeast, dispersing and adding into the materials, and stirring uniformly.

3. Peanut shell fermentation culture

After the fermentation material is inoculated, firstly stacking, inserting a thermometer, suspending and paving a plastic film, carrying out heat preservation and moisture preservation culture fermentation, when the temperature of the material rises to 35-40 ℃, spreading and paving, controlling the thickness of a material layer to be 10-20cm, suspending and paving the plastic film, and continuing culture. In the culture process, according to the culture temperature requirement, the materials are intermittently stirred and cooled, or the thickness of the material layer is adjusted, and the material temperature is controlled to be 30-45 ℃. The culture was carried out for 5 days. Making into peanut shell preparation 1. As shown in fig. 2.

Example 4

The ingredients were the same as in example 3. Weighing 20kg of seed koji according to the inoculation amount of 20%, adding into the materials, and stirring uniformly. The culture method was the same as in example 3. Culturing for 3 days to obtain peanut shell preparation 2. .

Embodiment 5 this embodiment relates to the detection of peanut shell preparation to contaminated soil heavy metal adsorption effect:

the peanut shell preparations fermented by the bacillus licheniformis in the examples 3 and 4 are respectively added into the polluted soil according to the proportion of 1 percent. Aging for 12 days. The leaching amount of heavy metal ions leached from the soil is detected, and the contaminated soil is treated under the same conditions by using uncultured peanut shells as a control, and the measurement results are shown in table 1. As shown in table 1: after the peanut shells are fermented by the bacillus licheniformis, the adsorption quantity of chromium in the polluted soil is improved from 88.4 percent to 100 percent; the adsorption quantity of cadmium is improved from 41.34 percent to 98 percent; the lead adsorption amount is improved from 68.14 percent to 94 percent; the adsorption amount of nickel is improved from 20.14 percent to 63 percent. The adsorption amount of copper is improved from 65.6 percent to 95.7 percent. Therefore, the adsorption capacity of the peanut shells to various heavy metals is obviously improved after the peanut shells are fermented by the bacillus licheniformis strain.

TABLE 1 influence of fermented peanut Shell on the treatment of contaminated soil on the leaching amount of heavy Metal elements

While the adsorption capacity of the bacillus licheniformis and bacillus licheniformis fermented peanut shells to the heavy metal in the polluted soil is inspected, the change of the cadmium and mercury effective state content in the polluted soil treated by the bacillus licheniformis and bacillus licheniformis fermented peanut shells is also inspected, and the result is shown in table 2. As shown in table 2: the peanut shells are fermented by the bacillus licheniformis strain, so that the effective state contents of cadmium and mercury in the soil can be obviously reduced. Compared with the unfermented peanut shells, the reduction of the content of the cadmium effective state in the polluted soil is improved from 51.30% to 96.47%; the reduction of the effective state content of mercury is improved from 52.38% to 90.47%;

table 2. influence of Bacillus licheniformis fermentation of peanut shells on the effective state of Cd and Hg in soil in treating contaminated soil

The above results illustrate that: the peanut shells are fermented by the bacillus licheniformis strain capable of producing pectinase, so that the capability of the peanut shells in repairing heavy metal polluted soil can be obviously improved. The method has the advantages of effective utilization of peanut shells and important social significance and economic value for repairing polluted environment.

1. the invention hydrolyzes the pectin in the peanut shell by utilizing the biotechnology, so that more oxygen-containing groups (such as hydroxyl, carboxyl, carbonyl and the like) are exposed; aldehyde groups and carboxyl groups generated by pectin hydrolysis are increased; organic acid generated by microbial metabolism, and loosening the structure of the peanut shell, and increasing the contact area of oxygen-containing groups and adsorbed heavy metals, thereby improving the passivation capacity of the peanut shell on the heavy metals from multiple aspects;

2. the method has the advantages of simple operation, less equipment investment, wide raw material source, low price, waste utilization, reduction of incineration pollution and popularization deserving.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims

1. A Bacillus licheniformis, characterized in that the Bacillus licheniformis (B), (B)Bacillus licheniformis) Is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 20977.

2. A biological agent produced from bacillus licheniformis fermented peanut shells according to claim 1.

3. A method of preparing a biological agent as claimed in claim 2, comprising the steps of:

inoculating the bacillus licheniformis of claim 1 into a fermentation medium for fermentation; the fermentation medium comprises peanut shells;

the preparation method of the fermentation medium comprises the following steps:

crushing peanut shells, adding the crushed peanut shells into the nutrient solution, and uniformly dispersing;

the fermentation temperature is 30-45 ℃.

4. The method for preparing a biological agent according to claim 3, wherein the edible alkali, the dipotassium hydrogen phosphate and the magnesium sulfate are respectively present in the nutrient solution in a mass fraction of 2%, 0.1% and 0.02%, and the weight ratio of the peanut shell to the nutrient solution is 1: (1-1.2).

5. The method of claim 3, wherein the ratio of the amount of Bacillus licheniformis to the amount of fermentation medium is: 1-3 eggplant bottle seeds are inoculated into every 10kg of peanut shells.

6. Use of the biological agent of claim 2 in the treatment of heavy metal ions in soil.